Activity of selected phytochemicals against Plasmodium falciparum.

According to the WHO, in 2008, there were 247 million reported cases of malaria and nearly one million deaths from the disease. Parasite resistance against first-line drugs, including artemisinin and mefloquine, is increasing. In this study the plant-derived compounds aglafolin, rocaglamid, kokusaginine, arborine, arborinine and tuberostemonine were investigated for their anti-plasmodial activity in vitro. Fresh Plasmodium falciparum isolates were taken from patients in the area of Mae Sot, north-western Thailand in 2008 and the inhibition of schizont maturation was determined for the respective compounds. With inhibitory concentrations effecting 50%, 90% and 99% inhibition (IC(50), IC(90) and IC(99)) of 60.95 nM, 854.41 nM and 7351.49 nM, respectively, rocaglamid was the most active of the substances, closely followed by aglafoline with 53.49 nM, 864.55 nM and 8354.20 nM. The activity was significantly below that of artemisinin, but moderately higher than that of quinine. Arborine, arborinine, tuberostemonine and kokusaginine showed only marginal activity against P. falciparum characterized by IC(50) and IC(99) values higher than 350 nM and 180 µM, respectively, and regressions with relatively shallow slopes S>14.38. Analogues of rocaglamid and aglafoline merit further exploration of their anti-plasmodial activity.

Thrombocyte counts in mice after the administration of papaya leaf suspension.

Following up a popular use of crude leaf preparations from Carica papaya for the treatment of dengue infections, a suspension of powdered Carica papaya leaves in palm oil has been investigated for its effect on thrombocyte counts in mice, administering by gavage 15 mg of powdered leaves per kg body weight to 5 mice. Equal numbers of animals received corresponding volumes of either palm oil alone or physiological saline solution. Thrombocyte counts before and at 1, 2, 4, 8, 10, 12, 24, 48 and 72 hours after dosing revealed significantly higher mean counts at 1, 2, 4, 8, 10 and 12 after dosing with the C. papaya leaf formulation as compared to the mean count at hour 0. There was only a non-significant rise of thrombocyte counts in the group having received saline solution, possibly the expression of a normal circadian rhythm in mice. The group having received palm oil only showed a protracted increase of platelet counts that was significant at hours 8 and 48 and obviously the result of a hitherto unknown stimulation of thrombocyte release. The results call for a dose-response investigation and for extending the studies to the isolation and identification of the C. papaya substances responsible for the release and/or production of thrombocytes.

Activity of Eurycoma longifolia root extract against Plasmodium falciparum in vitro.

The habitats of Eurycoma longifolia Jack, a slender tree, are jungles in Malaysia and Indonesia. It belongs to the family Simaroubaceae and is a source of quassinoids with anabolic, antimalarial and cytostatic activity. In this study, conducted during 2008 in Mae Sot, Thailand, a standardized extract of E. longifolia containing three major quassinoids, eurycomanone (1), 13,21-dihydroeurycomanone (2) and 13alpha(21)-epoxyeurycomanone (3) was evaluated for antiplasmodial activity against Plasmodium falciparum and its activity has been compared with that of artemisinin, using 38 fresh parasite isolates and assessment of inhibition of schizont maturation. The IC(50), IC(90) and IC(99) values for artemisinin were 4.30, 45.48 and 310.97 microg/l, and those for the root extract from E. longifolia 14.72, 139.65 and 874.15 microg/l respectively. The GMCOC for artemisinin was 337.81 mug/l, and for the plant extract it was 807.41 microg/l. The log-concentration probit regressions were parallel. The inhibitory activity of the E. longifolia extract was higher than that expected from the three quassinoids isolated from the plant, suggesting synergism between the quassinoids or the presence of other unidentified compounds.

Pharmacodynamic interaction between 4-aminoquinolines and retinol in Plasmodium falciparum in vitro.

The pharmacodynamic interaction between retinol and 4-aminoquinolines has been investigated in 29 fresh isolates of Plasmodium falciparum. Although the parasites were highly resistant against 4-aminoquinolines, significant synergism was observed between chloroquine and retinol as well as amodiaquine and retinol, the latter at physiological concentrations. Combination with retinol reduced the geometric mean concentrations effecting complete inhibition (GMCOC) by chloroquine from 14425 nM to 8943 nM in CHL-RET low, 7042 nM in CHL-RET medium, and 4920 nM in CHL-RET high. Synergism between amodiaquine and retinol was greater, with strong and highly significant reductions of the GMCOC, from 2520 nM for amodiaquine to 1092 nM for AMO-RET low, 800 nM for AMO-RET medium, and 745 nM for AMO-RET high. While it is obviously too late for making practical use of the activity enhancement for chloroquine, the situation is different for amodiaquine, where supplementation with retinol may extend the usefulness of the medicament.

Synergistic interaction between atovaquone and retinol in Plasmodium falciparum in vitro.

The study has been conducted with the objective of assessing the blood schizontocidal activities of atovaquone (ATO), retinol (RET) and combinations of both (ATO-RET) at set retinol concentrations corresponding to the 50th, 65th and 80th percentile of the physiological serum retinol levels. The in vitro tests followed the WHO standard protocol Mark II for measuring the inhibition of schizont maturation in Plasmodium falciparum. Valid results for all 5 test lines were obtained with 26 fresh parasite isolates from northwestern Thailand, an area affected by multidrug-resistance. The EC(50) values for atovaquone, retinol and for ATO in ATO-RET low, medium and high were 3.1 nM, 561.8nM, 0.85 nM, 0.73 nM and 0.45 nM, respectively, the EC(90) values 33.7 nM, 9338.6 nM, 25.31 nM, 8.89 nM, and 5.42 nM. The geometric mean cut-off concentrations of schizont maturation of atovaquone alone and for atovaquone in ATO-RET low, medium and high were 282.5 nM, 79.0 nM, 38.7 nM and 23.7 nM, respectively. These results and those of the Berenbaum analysis based on the fractional inhibitory concentrations indicate synergistic pharmacodynamic interaction between atovaquone and retinol, a phenomenon suggesting that the antimalarial activity of atovaquone could be enhanced by supplementation with retinol.

Development of a pharmacodynamic screening model with Crithidia fasciculata.

The genus Crithidia is a member of the family Trypanosomatidae and is related to the genera Leishmania and Trypanosoma with which it shares a variety of biochemical mechanisms, such as polyamine synthesis and methionin salvage. In consequence, a screening system for antiparasitic candidate material has been developed with Crithidia fasciculata, a parasite naturally occurring in insects and amphibians, but devoid of pathogenicity for humans. Initially a variety of culture media were evaluated of which TPS was best suited for the maintenance of stock cultures, and E-medium - a newly developed formula - for sensitivity testing. Optimal growth of C. fasciculata was observed under microaerophilic conditions. A system for sensitivity testing was developed and applied to the investigation of extracts from higher tropical plants of the genera Stemona and Aglaia for anticrithidial activity. Extracts with significant anti-crithidial activity were scheduled for chromatographic fractionation and the subsequent isolation, purification and structural identification of individual compounds for further sensitivity testing. Encouraging results were obtained with extracts from Aglaia odorata leaves, A. elaeagnoidea stem bark and A. edulis leaves, with EC(90) values of 1213 ng/ml, 1606 ng/ml, and 1462 ng/ml, respectively.

Synergism of desbutyl-benflumetol and retinol against Plasmodium falciparum in vitro.

This in vitro study was conducted to assess the blood schizontocidal activity of desbutyl-benflumetol (DBB), a new benzindene derivative, retinol and a combination of both compounds. The tests were carried out according to the methodology of the WHO standard test Mark II, measuring the drug-dependent inhibition of schizont maturation, and using 43 fresh isolates of Plasmodium falciparum from northwestern Thailand, an area with established multidrug-resistance. DBB and retinol showed a mean 50% effective concentration (EC-50) of 3.73 nM and 466.86 nM and 90% effective concentration (EC-90) of 19.83 nM and 5531.69 nM respectively. The combination of DBB and 3.50 muM retinol showed strong inhibition of schizont maturation, with an EC-90 for DBB of 0.67 nM. At the therapeutically relevant EC-99, the combination was about 10 times more effective than expected, suggesting that retinol potentiated the effect of DBB. A concentration of 3.50 muM retinol corresponds to the 95th percentile of the physiological serum levels. It is well known that retinol levels are significantly decreased in acute falciparum malaria. Supplementation with retinol during malaria treatment may improve the therapeutic results of blood schizontocides of the fluorene class.

Coartemether (artemether and lumefantrine): an oral antimalarial drug.

Coartemether (Riamet, Coartem, Novartis), a tablet formulation of artemether and lumefantrine, is a well-tolerated, fast-acting and effective blood schizontocidal drug that serves primarily in the treatment of uncomplicated falciparum malaria that is resistant to other antimalarials. Initial clinical-parasitological response relies mainly on the artemether component, while lumefantrine effects radical cure. The absorption of lumefantrine is poor during the fasting state, the normal condition in acutely ill malaria patients, but with return to normal diet it becomes adequate. This highlights the need for an appropriate adjustment of the dose regimen. In the area where Plasmodium falciparum shows the highest degree of multidrug resistance worldwide, the best results (99% cure) were obtained with a six-dose regimen given over 5 days. Extensive cardiological investigations have demonstrated the high cardiac safety of coartemether.

In-vitro response of Plasmodium falciparum to the main alkaloids of Cinchona in northwestern Thailand.

The blood schizontocidal activity of the four main Cinchona alkaloids against Plasmodium falciparum was compared in 46 fresh parasite isolates, using an in-vitro test measuring the drug-specific inhibition of schizont maturation. The studies were conducted in June-August 2001 at Mae Sot, northwestern Thailand, an area where quinine alone is no longer able to eliminate infections with P. falciparum. Quinidine showed the highest blood schizontocidal activity, followed by cinchonine, cinchonidine and finally quinine, which was identified as the least active compound. The isolates showed marked heterogeneity in their response to the Cinchona alkaloids. There was also high correlation of activity among all four alkaloids. The mean EC50 values for quinine, quinidine, cinchonine and cinchonidine were 144 nM, 80 nM, 104 nM and 225 nM, respectively, and the EC99 values 8040 nM, 861 nM, 1176 nM and 6531 nM. The EC99 values for quinine and cinchonidine are beyond the therapeutic concentration range and those for quinidine within it. For cinchonine, values are likely to be within this range, but toxicological and pharmacokinetic studies on this compound are required for clarifying its potential future role in the treatment of falciparum malaria.

Histidine-rich protein II: a novel approach to malaria drug sensitivity testing.

The production of histidine-rich protein II (HRP2), a histidine- and alanine-rich protein produced by Plasmodium falciparum, is closely associated with the development and proliferation of the parasite and therefore is perfectly suited to reflect growth inhibition as a measure of drug susceptibility. It was the aim of the present study to develop a malaria drug sensitivity assay based on the measurement of HRP2 in a simple enzyme-linked immunosorbent assay (ELISA). The new test proved to be as reliable as traditional in vitro assays, while it was considerably easier to establish and perform. Parasites are incubated at an initial level of parasitemia of 0.01 to 0.1% on microculture plates predosed with ascending concentrations of antimalarial drugs. After incubation for 48 to 72 h, the samples are freeze-thawed and transferred to ELISA plates. The complete ELISA takes about 2.5 h to perform, may be carried out with commercially available test kits, and requires relatively little technical equipment. In correlation analysis, the results closely paralleled those obtained by the isotopic assay (R = 0.892; P < 0.0001) and World Health Organization schizont maturation tests (R = 0.959; P < 0.0001). The novel HRP2 drug susceptibility assay proved to be very sensitive, simple to establish, and highly reproducible. It can be used for a wide range of applications, from epidemiological studies to the screening of new drugs, and may have the potential to replace traditional in vitro techniques. Standard operating procedures, updated information, and analytical software are available from http://malaria.farch.net.